Invented nearly 400 years ago, the optical microscope still remains one of the most powerful experimental tools in all of science and technology. Although diffraction limits the size of the smallest features one expects to resolve to ˜λ/2, i.e. ˜250 nm for visible light, recently a new arsenal of techniques has emerged that allow fluorescence imaging with resolution below this fundamental limit. These approaches are based on the realization that fluorescent objects in close proximity can still be resolved if their emission is separated by wavelength, time, or fluorescence lifetime or by directly reducing the size of the point-spread-function (PSF) of the microscope. The ability to image below the diffraction limit has already enabled researchers to, for example, decipher the coordination of the two heads of a molecular motor, bar-code DNA sequence information, measure the nano-scale distribution of proteins inside a cell, and track in real-time the movement of small organelles.
Many biological processes involve the coordinated action of several components that assemble in large protein complexes. Their size (˜10's of nm) and transient nature precludes studies of these complexes with conventional structural biology techniques such as single-particle reconstruction by electron microscopy and x-ray crystallography. Fluorescence microscopy on a nm scale could in principle enable real-time imaging of the position of single-molecules in physiological conditions with resolution rivaling that of electron microscopy. The resolution demonstrated to date is typically in the range of 10-20 nm, well above the size (˜5 nm) of a typical protein molecule.
Two previous studies addressed the problem of co-localizing single molecules in simultaneous two-color wide-field fluorescence imaging. In “Fluorescence imaging for monitoring the colocalization of two single molecules in living cells,” Biophys. J. 88, 2126-36, Koyama-Honda et al. teach that bright field imaging of a micro-fabricated array of 1 μm holes in a metal film, spaced by 5 μm, can be used to produce an overlay of two color images to within 20-30 nm. In “Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time,” Proc. Nat. Acad. Sci. 102, 1419-23, Churchman et al. teach that co-localization to ˜10 nm can be achieved by tracking a fluorescent bead that appears in two color images and that is translated by 0.5 μm on a grid pattern between successive images.